Throughout this application various publications are referenced, many in parenthesis. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
The proteinase-activated receptors (PAR) are a family of four G-protein coupled receptors (Vu et al. 1991; Nystedt et al. 1994; Nystedt et al. 1995; Ishihara et al. 1997; Xu et al. 1998; Kahn et al. 1998) that share a unique mechanism of activation. Serine proteases such as trypsin and thrombin cleave PARs, unmasking an extracellular N-terminal domain that subsequently binds and activates the receptor, thus acting as a tethered ligand. Synthetic activating peptides that correspond to the tethered ligands of the various PARs can also activate their individual receptor directly (Al-Ani et al. 1999; Hollenberg et al. 1997).
The first PAR discovered was the thrombin receptor (protease activated receptor-1; PAR-1) from human platelet progenitor cells. The serine protease α-thrombin cleaves the extracellular, N-terminal peptide chain of PAR-1 between Arg-41 and Ser-42 to expose a truncated N terminus bearing the peptide recognition motif SEQ ID NO:5: SFLLRN. Synthetic peptides containing this epitope have full PAR-1 agonist properties independent of thrombin activation, confirming that the receptor-linked peptide sequence serves as an activating ligand. Notably, PAR-1 mediates most of the cellular actions of thrombin, including platelet aggregation, cell proliferation, inflammatory responses, and neurodegeneration.
Researchers have identified three more members of this class of G protein-coupled receptors, PAR-2, PAR-3, and PAR-4, which also are activated by a serine protease to initiate an intramolecular ligand-activation mechanism.
Nystedt et al. (1995) described the receptor they designated proteinase activated receptor-2 (PAR-2) as a member of the large family of 7-transmembrane-region receptors that couple to guanosine-nucleotide-binding proteins. Proteolytic cleavage of PAR-2's extracellular amino terminus leaves the new amino terminus, a tethered ligand, free to interact with another region of the receptor to effect receptor activation. Nystedt et al. (1995) noted that PAR2 shares this special mode of activation with the thrombin receptor, for which this mechanism was first described (see above).
Nystedt et al. (1994) cloned the mouse Par2 sequence from genomic DNA. When expressed in frog oocytes, the receptor could be activated with nanomolar concentrations of the serine protease trypsin but not with thrombin in doses up to 100 nM. It was judged that the gene was present in the genome in a single copy. Two protein-encoding exons were identified separated by 10 kb.
Nystedt et al. (1995) cloned the human gene. The deduced protein sequence was similar to that of the mouse Par2 receptor and, when expressed in Chinese hamster ovary cells, the human PAR2 responded to trypsin and a peptide from the receptor sequence. Northern blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine, and colon. Moderate expression was detected in many organs, but none in brain or skeletal muscle. By fluorescence in situ hybridization, the gene was mapped to 5q13. Nystedt et al. (1995) noted that the location of the thrombin receptor gene (F2R), also mapped to 5q13, which raised questions concerning the evolution of proteinase activated receptors.
The general structure of proteinase activated receptors is shown in FIG. 6. Referring to FIG. 6, the receptor (12) includes an amino terminus (18). Within the amino terminus (18) of the receptor (12) is a portion (20) which is a peptide agonist for the receptor. When the receptor is exposed to it's appropriate cleavage agent, the cleavage agent cleaves the most amino terminal part of the molecule (22) leaving the portion which is a peptide agonist (20) exposed. The peptide agonist (20) reacts with the binding site (24) for the peptide agonist (which is a part of the remainder of the receptor molecule) and binds thereto, activating the receptor. The site between the amino acid residues of the peptide agonist (20) and the most amino terminal part of the molecule (22) is the cleavage site (26).
The amino acid sequence of human PAR-2 is provided at GenBank Accession No. P55085. The receptor consists of 397 amino acids, of which 1-25 represent a signal peptide. Amino acids 26-36 represent a propeptide which is removed for receptor activation. Trypsin cleaves the receptor at SKGR ↓SLIG (the cleavage site; amino acids 33-40), leaving a peptide agonist (tethered ligand) having the amino acid sequence SEQ ID NO:6: SLIGKV-NH2 (amino acids 37-42). The residues of this peptide agonist interact with extracellular domains of the cleaved receptor, resulting in activation of the receptor. Steinhoff et al. (2000), Lerner et al. (1996), Kawabata et al. (1999), Andrade-Gordon et al. (1999), Bohm et al. (1996), Kahn et al. (1996), Dery et al. (1998), Nystedt et al. 1996, U.S. Pat. No. 6,017,890 (issued Jan. 25, 2000), PCT International Publication No. WO 99/42475 (published Aug. 26, 1999), and PCT International Publication No. WO 98/34948 (published Feb. 5, 1998), discuss protease activated receptors further, including the design of agonists and antagonists for these receptors, and are each incorporated herein by reference.
Recently, proteinase-activated receptor 2 (PAR-2) expression was detected on a subset of peripheral peptidergic neurons and was shown to be involved in the neurogenic component of inflammation (Steinhoff et al. 2000).
Inflammation and its sequelae are known to cause significant changes in the somatic sensory nervous system affecting peripheral nerves, spinal cord and supraspinal structures. Several biological agents induced by inflammation such as neurotrophins (e.g. nerve growth factor or NGF), prostanoids, bradykinin and various cytokines can sensitize peripheral nociceptors (Dray 1995). This leads to an increase in afferent signaling which in turn may cause sensitization at the central level, resulting in amplification and persistence of pain. Amongst the many potential sensitizing factors in this inflammatory soup, little attention has been paid to the role of proteases. These enzymes are derived from humoral (thrombin, factor Xa) and cellular (trypsin, tryptase and other tryptic enzymes from mast cells) sources (Cocks and Moffat 2000). In addition, inflammation in specific organs such as the pancreas may result in a significant release and activation of endogenous proteases (Hofbauer et al. 1998; Nguyen et al. 1999).
Pain associated with inflammation, or not, plaques many people. Any means for alleviating such pain would be useful.